Project Summary Human ASH1L (absent, small, or homeotic discs like 1) mediates proliferation and survival of hematopoietic stem cells and is often upregulated in leukemias. It is required for hematopoietic development and expression of developmental genes, including the HOX gene family. Upregulated activity of ASH1L, found in mixed lineage leukemia (MLL)-rearranged acute lymphoblastic leukemia (ALL), is generally associated with a poor prognosis. ASH1L is a major methyltransferase that methylates histone H3, generating the epigenetic mark H3K36me2 associated with transcriptional activation and elongation. ASH1L contains a unique combination of the catalytic methyltransferase SET domain and adjacent bromodomain (BD), a PHD finger, and a BAH domain with unclear biological roles. Our recent studies reveal that the BD, PHD and BAH domains of ASH1L are epigenetic readers capable of recognizing distinctive states of histone H3. The molecular mechanisms underlying these novel functions of ASH1L are unknown and will be elucidated in the proposed studies. We hypothesize that the concomitant recognition of distinct histone states by the PHD, BD and BAH domains recruits or stabilizes ASH1L at promoters of ASH1L target genes and is necessary for the catalytic activity of ASH1L and methylation of H3K36 at these genes. We seek to understand a crosstalk between the BD, PHD and BAH domains of ASH1L and determine the molecular mechanism and functional significance of the multivalent engagement of ASH1L with chromatin. We will employ complementary in vitro and in vivo approaches to establish the molecular and structural basis and define the biological importance of histone binding by ASH1L readers. This research will provide atomic-resolution insights into ASH1L signaling pathways that may constitute new targets for therapeutic interventions and enhance our knowledge of fundamental principles underlying the epigenetic-driven gene transcription. It will also lead to a better understanding of human cancers associated with aberrant activity of ASH1L, including acute leukemias.